2.1. Protamines
Protamines are small highly basic nucleoproteins associated with DNA that occur in no cells other than spermatids and spermatozoa. Those proteins are synthesized de novo and incorporated into the nuclei of spermatogenic cells during spermiogenesis where they replace histones and are bound to the spermatozoal DNA, a progression that takes place in the immunologically sequestered lumena of the seminiferous tubules of the postpuberal testis (Gilula, N. B., et al., 1976, Dev. Biol. 50:142).
Protamine 1 (originally termed p15) is a human protamine that occurs in four variants. It was identified and characterized by use of a monoclonal antibody (termed HPmAb, described infra) (Rodman, T. C., et al., 1983, Gamete Res. 8:129-147).
Human protamine 2 has long been recognized and known to occur as two variants (Kolk, A. H. and Samuel, T., 1975, Biochim. Biophys. Acta 393:307-319), later designated as protamine 2a and 2b (McKay, D. J., et al., 1986, Eur. J. Biochem. 156:5-8).
Polyvalent rabbit antisera specific for the two protamines of mouse sperm chromatin have been isolated and used to characterize the protamines as to distribution and DNA association, and to recognize immunochemical cross-reactivity among mammalian protamines (Rodman, T. C., et al., 1984, Exp. Cell Res. 150:269-281).
Polyclonal rabbit antisera against human protamines have been used in immunohistochemical studies of histone to protamine transition during spermiogenesis (Roux, Ch., et al., 1988, J. Reprod. Fert. 82:35-42). The detection steps of the immunoassays in this study were carried out by use of antibodies against rabbit IgG.
Mouse monoclonal antibodies to human protamines of the IgG1 and IgG2b isotypes have been isolated and partially characterized (Stanker, L. H., et al., 1987, Hybridoma 6(3):293-303). None of the described antibodies reacted with polyarginine.
Autoantibodies against spermatozoal antigens have been detected in vasectomized males, by Western blotting procedures assaying for antibodies reactive with spermatozoal antigens separated by SDS-polyacrylamide gel electrophoresis, or by agglutination tests (Hald, J., et al., 1987, J. Reprod. Immunol. 10:15-26). Since protamines are too basic to migrate in SDS-polyacrylamide gels, and have immunoreactive sites which are not accessible unless the chromatin is decondensed (Rodman, T. C., et al., 1970, J. Cell Biol. 80:605-620), the reported assays did not detect anti-protamine antibodies.